Recent developments in the pharmaceutical industry and in combinatorial chemistry have exponentially increased the number of potentially useful compounds, each of which must be characterized in order to identify their active components and/or establish processes for their synthesis. To more quickly analyze these compounds, researchers have sought to automate analytical processes and to implement analytical processes in parallel.
Various chemical and biochemical fluid phase separation processes are known, including chromatographic, electrophoretic, electrochromatographic, immunoaffinity, gel filtration, and density gradient separation. Each of these processes is capable of separating species in fluid samples with varying degrees of efficiency to promote their analysis.
One particularly useful fluid phase separation process is chromatography, which may be used with a wide variety of sample types and encompasses a number of methods that are used for separating ions or molecules that are dissolved in or otherwise mixed into a solvent. Liquid chromatography “LC”) is a physical method of separation wherein a liquid “mobile phase” (typically consisting of one or more solvents) carries a sample containing multiple constituents or species through a separation medium or “stationary phase.” Various types of mobile phases and stationary phases may be used. Stationary phase material typically includes a liquid-permeable medium such as packed granules (particulate material) disposed within a tube (or other channel boundary). The packed material contained by the tube or similar boundary is commonly referred to as a “separation column.” High pressure is often used to obtain a close-packed column with a minimal void between each particle, since better resolution during use is typically obtained from more tightly packed columns. As an alternative to packed particulate material, a porous monolith or similar matrix may be used. So-called “high performance liquid chromatography” “(HPLC”) refers to efficient separation methods that are typically performed at high operating pressures.
Typical interactions between stationary phases and solutes include adsorption, ion-exchange, partitioning, and size exclusion. Examples of types of stationary phases to support such interactions are solids, ionic groups on a resin, liquids on an inert solid support, and porous or semi-porous inert particles, respectively. Commonly employed base materials include silica, alumina, zirconium, or polymeric materials. A stationary phase material may act as a sieve to perform simple size exclusion chromatography, or the stationary phase may include functional groups (e.g., chemical groups) to perform other (e.g., adsorption or ion exchange separation) techniques.
Mobile phase is forced through the stationary phase using means such as, for example, one or more pumps, gravity, voltage-driven electrokinetic flow, or other established means for generating a pressure differential. After sample is injected into the mobile phase, such as with a conventional loop valve, components of the sample will migrate according to interactions with the stationary phase and the flow of such components are retarded to varying degrees. Individual sample components may reside for some time in the stationary phase (where their velocity is essentially zero) until conditions (e.g., a change in solvent concentration) permit a component to emerge from the column with the mobile phase. In other words, as the sample travels through voids or pores in the stationary phase, the sample may be separated into its constituent species due to the attraction of the species to the stationary phase. The time a particular constituent spends in the stationary phase relative to the fraction of time it spends in the mobile phase will determine its velocity through the column. Following separation in an LC column, the eluate stream contains a series of regions having an elevated concentration of individual component species. Thus, HPLC acts to provide relatively pure and discrete samples of each of the components of a compound. Gradient separations using conventional HPLC systems are typically performed within intervals of roughly five to ten minutes, followed by a flush or rinse cycle before another sample is separated in the same separation column.
Following chromatographic separation in a column (or other fluid phase separation), the resulting eluate (or effluent) stream contains a series of regions having elevated concentrations of individual species, which can be detected by various flow-through techniques including spectrophotometric (e.g., UV-Visible absorption), fluorimetric, refractive index, electrochemical, or radioactivity detection. Fluid phase separation with flow-through detection generally provides signal response that is proportional to analyte amount or concentration. As a result, fluid phase separations are often well-suited for quantitative analyses, but less suited for identifying or characterizing individual components-particularly when novel or previously uncharacterized compounds are used.
To provide increased throughput, parallel fluid phase separation systems including multi-column LC separation systems and multi-channel electrophoretic separation systems have been developed.
Another important analytical technique that can complement fluid phase separation is mass spectrometry “MS”), a process that analyzes ions utilizing electromagnetic fields. More specifically, MS permits molecular mass to be measured by determining the mass-to-charge ratio “m/z”) of ions generated from target molecules. A system for performing mass spectrometry typically includes an ionization source that generates ions from a sample and delivers them into the gas phase, one or more focusing elements that facilitate ion travel in a specific direction, an analyzer for separating and sorting the ions, and a transducer for sensing the ions as they are sorted and providing an output signal, along with vacuum pumping means and a vacuum enclosure surrounding at least the focusing elements and analyzer. MS is a fast analytical technique that typically provides an output spectrum displaying ion intensity as a function of m/z. One benefit of using MS is that it can provide unique information about the chemical composition of the analyte—information that is much more specific than that can be obtained using flow-through detection technology typically employed with most fluid phase separation processes. The ability to qualitatively identify molecules using MS complements the quantitative capabilities of fluid phase separations, thus providing a second dimension to the analysis.
Various mass spectrometric techniques are known, including time-of-flight “TOF”), quadrupole, and ion trap. In a TOF analyzer, ions are separated by differences in their velocities as they move in a straight path toward a collector in order of increasing mass-to-charge ratio. In a TOF MS, ions of a like charge are simultaneously emitted from the source with the same initial kinetic energy. Those with a lower mass will have a higher velocity and reach the transducer earlier than ions with a higher mass. In a quadrupole device, a quadrupolar electrical field (comprising radiofrequency and direct-current components) is used to separate ions. An ion trap (e.g., quadrupole-based) can trap ions and separate ions based on their mass-to-charge ratio using a three-dimensional quadrupolar radio frequency electric field. In ion trap instruments, ions of increasing mass-to-charge ratio successively become unstable as the radio frequency voltage is scanned.
Various conventional ionization techniques may be used with mass spectrometry systems. One prevalent technique is electrospray ionization (ESI), which is a “soft” ionization technique. That is, ESI does not rely on extremely high temperatures or extremely high voltages to accomplish ionization, which is advantageous for the analysis of large, complex molecules that tend to decompose under harsh conditions. In ESI, highly charged droplets of analyte dispersed from a capillary in an electric field are evaporated, and the resulting ions are drawn into a MS inlet. Other known ionization techniques include: chemical ionization (which ionizes volatilized molecules by reaction with reagent gas ions); field ionization (which produces ions by subjecting a sample to a strong electric field gradient); spark-source desorption (which uses electrical discharges or sparks to desorb ions from samples); laser desorption (which uses a photon beam to desorb sample molecules); matrix-assisted laser desorption ionization or “MALDI” (which produces ions by laser desorbing sample molecules from a solid or liquid matrix containing a highly UV-absorbing substance); fast atom bombardment or “FAB” (which uses beams of neutral atoms to ionize compounds from the surface of a liquid matrix); and plasma desorption (which uses very high-energy ions to desorb and ionize molecules in solid-film samples).
By coupling the outputs of one or more fluid phase separation process regions to a MS instrument, it becomes possible to both quantify and identify the components of a sample. There exist challenges, however, in providing efficient integrated fluid phase separation/MS systems. MS instruments are typically extremely complex and expensive to operate and maintain, due primarily to the need to precisely control the electromagnetic fields generated within such devices and the need to maintain vacuum conditions therein. Integrated fluid phase separation/MS systems including a single fluid phase process region coupled to a mass spectrometer instrument by way of an ESI interface are known, but they suffer from limited throughput since they can only analyze one sample at a time—and the upstream fluid phase separation process is typically much slower than the downstream mass analysis process. In other words, a fluid phase separation/MS analyzer system having only a single fluid phase separation process region fails to efficiently utilize the rapid analytical capabilities of the MS analyzer portion.
More efficient systems including multiple fluid phase separation process regions coupled to a single MS analyzer are also known and provide higher throughput compared to systems having only a single fluid phase separation process region, but these improved systems still suffer from limited utility. Examples are provided in U.S. Pat. No. 6,410,915 to Bateman, et al.; U.S. Pat. No. 6,191,418 to Hindsgaul, et al.; U.S. Pat. No. 6,066,848 to Kassel, et al.; and U.S. Pat. No. 5,872,010 to Karger, et al., each showing some variation of a multiplexed fluid phase (e.g., LC) separation/MS systems where the outputs of multiple simultaneously-operated fluid phase separation regions are periodically sampled by a single MS device. In these multiplexed systems, however, the MS can sample an effluent stream from only one fluid phase separation process region at a time. While one stream is being analyzed, the others must continue to flow, as these systems have no storage capacity. This inherently results in data loss. To mitigate this data loss, MS sampling must occur very quickly. The MS analyzer thus receives very small plugs of sample-containing effluent, reducing the ability of the MS instrument to integrate data in order to eliminate noise and resulting in reduced signal clarity. Additionally, such conventional systems typically utilize mechanical gating for directing desorbed effluent into a single MS inlet. Mechanical gating components limit the scalability and increase the complexity and cost of the resulting system.
Accordingly, there exists a need for improved analytical systems that permit parallel analysis of multiple samples. Advantageous system characteristics would include scalability to permit a large number of samples to be analyzed simultaneously at a relatively low cost per analysis with a minimal loss of data and/or signal clarity. Ideally, an improved system would be comparatively simple and inexpensive to build, operate, and maintain.